Digyalipopeptide A, an antiparasitic cyclic peptide from the Ghanaian Bacillus sp. strain DE2B

During the continued isolation of different bacteria from highly diverse, low human activity environments in Ghana and the subsequent characterization and biological activity studies of their secondary metabolites, we found both Gram-positive and Gram-negative Bacillus strains to be ubiquitous and widespread. One of such strains, the Ghanaian novel Bacillus sp. strain DE2B was isolated from rhizosphere soils collected from the Digya National Park in Ghana. Chromatographic purifications of the fermented culture extract of the strain DE2B, led to the isolation of a cyclic lipopeptide, digyalipopeptide A (1). Using 1D and 2D NMR data, mass spectrometry sequence tagging, advanced Marfey’s analysis, and the GNPS molecular networking we solved the full structure of digyalipopeptide A (1). We found that compound 1 is a member of a somewhat homologous series of peptides produced as a mixture by the strain containing the same amino acid sequence in the cyclic peptide backbone but differing only by the length of aliphatic fatty acid side chains. When tested against Trypanosoma brucei subsp. brucei strain GUTat 3.1 and Leishmania donovani (Laveran and Mesnil) Ross (D10), digyalipopeptide A (1) gave IC50 values of 12.89 µM (suramin IC50 0.96 µM) and 4.85 µM (amphotericin B IC50 4.87 µM), respectively. Furthermore, digyalipopeptide A (1) produced IC50 values of 10.07 µM (ampicillin IC50 0.18 µM) and 10.01 µM (ampicillin IC50 1.53 µM) for Staphylococcus aureus and Shigella sonnei, respectively. The selectivity and toxicity profile of compound 1 was investigated using normal cell lines, macrophages RAW 264.7. When tested against normal macrophages, compound 1 gave an IC50 value of 71.32 μM. Selectivity indices (SI) were obtained by calculating the ratio of the IC50 in RAW 264.7 to the IC50 in the respective microbe and neglected parasite. In the presence of RAW 264.7 cell lines, compound 1 was particularly selective towards Leishmania donovani (Laveran and Mesnil) Ross (D10) with an SI value of 14.71. The bioactivity studies conducted confirm the role of these cyclic lipopeptides as defense chemicals in their natural environment and their ability to be biologically active across different species.


Sediment sample collection site
The Ghanaian Bacillus sp. strain DE2B was isolated from soil sediments sampled within the Digya National Park (coordinates: 7 • 31'44.85'' N and 0 • 036'48.15'' W), a forest reserve which is home to a wide variety of organisms and located along the western coasts of the Lake Volta in the Bono-East region of Ghana. This sampling site is well noted for the predominance of many undisturbed natural habitats including wetlands which represent one of the most bio-diverse environments in the country.

Culture and isolation of strain DE2B from soil sediment
In a manner similar to the procedure from [1] the soil sediment coded DE2B was prepared for the isolation of bacteria strains by placing 5 g of the soil sample into a new sterile 50 mL falcon tube. About 10 mL of sterilized Milli Q water was added and the falcon tube was capped under sterile conditions. The sample was subsequently placed in a hot water bath at 55 °C for 1 hour to prevent growth of fast-growing gram-negative bacteria and fungi. The heated soil sample was allowed to cool and then placed in a clean bench which had been sterilized with 70% ethanol followed by exposure to ultraviolet (UV) light for an hour. About 10 mL of sterilized tap water was added to sample and dilutions of 10 −1 , 10 −2 and 10 −3 concentrations were prepared. This was done by pipetting 1 mL of the stock 10 mL suspension into a new sterile 50 mL falcon tube and subsequently diluting with 9 mL of sterilized tap water after which it was shaken thoroughly to afford the 10 −1 dilution. A 1 mL aliquot of the 10 −1 suspension was used to prepare the subsequent serial dilutions. Pre-sterilized inoculation loops were used to smear evenly about 5 µL of the different concentrations of the soil sample on pre-modified ISP2 agar plates at pH 5.5 supplemented with 25 μg/mL each of nystatin and nalidixic acid antibiotics. The master plates were parafilmed and incubated for 7 days at 28 °C to allow the growth of bacteria. After the 7-day period, the agar plates were taken out of the incubator and observed in a clean bench.
Single colonies with different phenotypes were re-plated on new agar plates using sterilized inoculation loops in the clean bench to obtain pure strains. Parent or master plates that had fungal contamination or did not have single individual colonies to pick were discarded to prevent contamination of the lot. The plates were sealed using parafilm and put in the incubator to allow for continued bacteria growth. Pure strains that had been transferred onto new agar plates were also parafilmed and placed in the incubator for periodic observation. Pure strains were obtained by successively sub-culturing all colonies originally sub-cultured from the parent or master plates. A pure strain of Bacillus sp. DE2B was obtained through this process and its whole DNA submitted to the Department of Biochemistry, Sanger Sequencing, University of Cambridge, UK for whole genome sequencing.

Identification of strain DE2B
Identification of the closest phylogenetic neighbor and calculations of pairwise 16S rRNA gene sequence similarities were achieved with the EzBioCloud web service by Yoon et al., 2017 [2].
Multiple sequence alignments were obtained using the CLUSTAL W application by Thompson, Higgins and Gibson, 1994 [3] and phylogenetic analysis was performed using the MEGA v.7.0 software package by Kumar et. al. in 2016 [4]. Evolutionary distance calculations were obtained using the Jukes-Cantor model [5] while Neighbor-joining was achieved using Saitou and Nei 1987 [6]. Maximum-parsimony, Felsenstein, 1983 [7] and Maximum-likelihood, Felsenstein, 1981 [8] methods were used to infer the phylogenetic tree. The resultant tree topologies were assessed by bootstrap analyses, Felsenstein, 1985 [9] based on 1000 re-samplings of the datasets.
The relative analysis of the 16S rRNA gene sequence of strain DE2B, revealed its phylogenetic association to the genus Bacillus, particularly, members of the Bacillus cereus group sharing over 98.05% similarity with the known species of this group, Figure S3 Table S1. Mass spectrometry data is detailed in figure S17 sodium phosphate monobasic (KH2PO4), sodium hydroxide (NaOH), and sodium bicarbonate (NaHCO3) were purchased from Sigma-Aldrich, USA.

Preparation of compound for bioassay
A stock solution of 10 mM concentration of compound 1 was prepared by first drying the compound using nitrogen gas and weighing on a mass balance (AND GH-120, A and D Company Limited, Tokyo, Japan) to determine the weight before dissolving in an appropriate volume of dimethyl sulfoxide (DMSO) to obtain the 10 mM concentration. Subsequently, the solution was vortexed (MSI Minishaker, IKA Company, Osaka, Japan) and filter sterilized into vials through 0.45 µm millipore filters under sterile conditions. The solution was then stored at −20 °C until used.

Cell culture
The GUTat 3.1 strain of the bloodstream form of T. brucei brucei parasites was used in this study. Parasites were cultured in vitro according to the conditions established previously by Yabu et al. in 1998 [12]. Parasites were used when they reached a confluent concentration of 1 × 10 6 parasites/mL. Estimation of parasitemia was done with the Neubauer counting chamber.
Parasites were diluted to a concentration of 3 × 10 5 parasites/mL with IMDM medium and used for the drug assay.
The log-phase promastigotes of L. donovani (D10) and L. major (NR48815) were cultured in M-119 growth medium with a working concentration of 6 × 10 6 cells/mL. Parasites were used when they reached a confluent concentration of 1 × 10 6 parasites/mL. Estimation of parasitemia was done with the Neubauer counting chamber. Parasites were diluted to a concentration of 3 × 10 5 parasites/mL with M199 medium and used for the drug assay [13].

In vitro viability test for trypanosomes and macrophages
The viability of the treated or untreated trypanosome parasites were ascertained by the Alamar Blue assay test in a manner similar to a procedure from [13]. The assay was carried out in a 96well plate through the instructions of the manufacturer with slight modifications. About 1.5 × 10 4 parasites were seeded with varied concentrations of the compound 1 ranging from 0 µM to 100 µM. Final concentrations of DMSO were maintained at 0.1%, respectively. After incubation of parasites with or without the compound for 24 hours at 37 °C in 5% CO2, 10% Alamar Blue dye was added, and the parasites were incubated for another 24 hours in darkness. A Tecan Sunrise Wako spectrophotometer was used to read the absorbance at 540 nm for the plate after 48 hours. Suramin was used as the control.
For normal macrophages RAW 264.7, cell lines were plated at a density of 3.0 × 10 5 cells/mL for 48 hours to allow for sufficient adherence to plates before adding the compound to the cells in a two-fold dilution and subsequent incubation for another 24 hours.

In vitro viability test for Leishmania parasites
As previously described in [14], the Alamar Blue assay was carried out on treated and untreated Leishmania parasites to ascertain their viability. The assay was performed in a 96-well plate following the manufacturer's instructions, with modification. About 3 × 10 5 parasites were seeded with varied concentrations of the compound ranging from 0 µM to 100 µM. Final concentrations of DMSO were kept at 0.1%. After incubation of parasites with or without the compound for 24 hours at 28 °C, 10% Alamar Blue dye was added, and the parasites were incubated for another 24 hours in darkness. After a total of 48 hours, the plate was read for absorbance at 540 nm using a Tecan Sunrise Wako spectrophotometer, AUSTRIA GmbH (Salzburg, Austria). The trend curve was drawn to obtain a 50% inhibitory concentration (IC50) for the compound.

In vitro viability test for laboratory microbes
In a manner similar to [1] Figure S3: Modified Kupchan solvent partitioning of the crude extract of Bacillus sp. DE2B gives FH, FD, FM, and WB fractions. Figure S4: Sephadex LH-20 chromatography of FD fraction followed by reversed phase semipreparative HPLC gives pure compound 1. S18 Figure S5: First HPLC chromatogram for FD-SFB.

Supplementary tables
*First HPLC chromatogram for FD-SFB produces the five-member homologous series of cyclic lipopeptides 1-5. Compound 1 is collected and re-injected on HPLC to further purify it for full structure elucidation.